Abstract Intracellular signaling pathways depend upon appropriate and unique subcellular locations of their constituent proteins. Frequently, key intracellular signaling proteins move from one subcellular location to another in response to extracellular stimuli. Incorrectly localized proteins can prevent completion of a particular signaling pathway or can cause unregulated signaling, contributing to disease states such as cancer and hypertrophy of the heart. Understanding how cellular proteins come together at specific times and subcellular sites will lead to insight into ways to block inappropriate signaling in disease. Mechanisms responsible for reversibly targeting peripheral membrane proteins to different cellular membranes are poorly understood. Moreover, it is not clear how different subcellular localizations influence a signaling protein's function. This application will focus on several key questions regarding the mechanisms and function of intracellular trafficking of heterotrimeric (a[unreadable]?) G proteins. G proteins act as molecular switches to relay information from cell surface receptors to appropriate effector proteins. To transmit a signal from a G protein-coupled receptor (GPCR), G proteins must localize to the cytoplasmic face of the plasma membrane (PM). However, it is becoming increasing clear that G proteins are not statically localized at the PM, but instead follows distinct trafficking pathways to reach the PM and internalize and recycle to the PM in response to GPCR activation. Additionally, new findings suggest that G proteins have important, yet poorly defined, signaling functions at subcellular locations other than the PM. To address these issues, the major objectives of this proposal are 1) Define pathways of endomembrane to PM trafficking of Ga and G[unreadable]?;2) Define trafficking pathways and recycling mechanisms of GPCR-activated Ga and G[unreadable]?;3) Define the role of G[unreadable]? in regulating a Golgi-restricted signaling pathway. This research will utilize cultured mammalian cells as model systems and will employ a number of techniques, including immunofluorescence microscopy, fluorescence microscopy of live cells, subcellular fractionation, and numerous biochemical assays to define structure-function relationships in terms of mechanisms of reversible G protein trafficking. Public Health Relevance: G protein-mediated signaling pathways regulate numerous physiological responses, including cardiovascular function, neurotransmitter responses, cell differentiation, cell migration, immune cell function, and smell, taste, and vision, and dysregulation of G protein-mediated signaling pathways contribute to numerous disease states, including heart disease, hypertension, cancer, metastasis, endocrine disorders, and blindness. Understanding trafficking of G proteins and how select signaling pathways are affected by differential localization of G proteins will provide an opportunity to therapeutically target unwanted G protein responses while leaving intact beneficial G protein signaling pathways.